Oncogene (1998) 16, 1125 ± 1130 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 Increased transversions in a novel mutator colon cancer cell line James R Eshleman1,6, P Scott Donover2, Susan J Littman5, Sandra E Swinler2, Guo-Min Li4,7, James D Lutterbaugh2, James KV Willson2, Paul Modrich4, W David Sedwick2, Sanford D Markowitz2 and Martina L Veigl3 1Departments of Pathology; 2Medicine; 3General Medical Sciences and Ireland Cancer Center, University Hospitals of Cleveland and Case Western Reserve University, Suite 200, UCRC II, 11001 Cedar Road, Cleveland, Ohio 44106; 4Department of Biochemistry and Howard Hughes Medical Institute; 5Department of Medicine-Oncology, Duke University Medical Center, Nanaline Duke Building, Durham, North Carolina, 27710 USA We describe a novel mutator phenotype in the Vaco411 In this report we describe the unique speci®city of a colon cancer cell line which increases the spontaneous new mutator defect, which is independent of MMR. mutation rate 10 ± 100-fold over background. This We have previously shown that the Vaco411 colon mutator results primarily in transversion base substitu- cancer cell line displays a moderately (10 ± 100-fold) tions which are found infrequently in repair competent increased spontaneous mutation rate (Eshleman et al., cells. Of the four possible types of transversions, only 1995), and does not exhibit the RER phenotype which three were principally recovered. Spontaneous mutations generally accompanies MMR defects (Eshleman and recovered also included transitions and large deletions, Markowitz, 1995). We now describe the spontaneous but very few frameshifts were recovered. When compared hprt mutations arising in this new colon cancer to known mismatch repair defective colon cancer mutator, and compare these mutations to the repair mutators, the distribution of mutations in Vaco411 is capacity exhibited by cell-free extracts of this cell line. signi®cantly dierent. Consistent with this dierence, The spontaneous mutant collection lacks frameshifts, Vaco411 extracts are pro®cient in assays of mismatch which are commonly seen in MMR defective cell lines repair. The Vaco411 mutator appears to be novel, and is (Eshleman et al., 1996; Bhattacharyya et al., 1995; Kat not an obvious human homologue of any of the et al., 1993). Instead, transversion-type base substitu- previously characterized bacterial or yeast transversion tions predominate, characterizing this cell line as a phenotypes. Several hypotheses by which this mutator novel transversion mutator. may produce transversions are presented. Keywords: colon cancer; HPRT; mutator phenotype; transversions Results Transversions are the most frequent spontaneous hprt mutation recovered from Vaco411 Introduction Individually isolated, spontaneous, 6-thioguanine Mutator phenotypes have been proposed as a (6TG) resistant mutants were initially characterized prerequisite for multi-step carcinogenesis (Nowell, by hprt RT ± PCR and cDNA sequencing (Table 1). 1976; Loeb, 1991). By increasing the mutation rate, a Base substitutions are the predominant type of mutator phenotype permits the multiple independent mutation recovered (70% of total mutations). Trans- mutations in oncogenes and tumor suppressor genes versions (50% of total mutations and 71% of base required for multi-step carcinogenesis. Direct evidence substitutions) occur more than twice as frequently as that mutators play an important role in carcinogenesis transitions (20% of total mutants and 29% of base is provided by the recent discovery of a complex substitutions). Of note, small (1 ± 4 bp) deletions, which mutator phenotype in colorectal cancer (CRC) are prevalent in MMR defective human cell lines, are resulting from defects in mismatch repair (MMR), see almost absent (only 5%) among the Vaco411 muta- reviews (Eshleman and Markowitz, 1996; Modrich and tions. Finally, 20% of the Vaco411 spontaneous hprt Lahue, 1996; Kolodner, 1995; Umar and Kunkel, mutants harbor large deletions that remove one or 1996). This mutator is characterized by a 100 ± 1000- more coding exons. fold increased spontaneous mutation rate in the hprt gene target (Eshleman et al., 1995; Bhattacharyya et Sequence context of transversions al., 1994; Branch et al., 1995). As discussed above, transversions predominate among the spontaneous mutations seen in Vaco411. A sequence level analysis of the transversions is depicted Correspondence: ML Veigl or WD Sedwick, Ireland Cancer Center Research Laboratories, Suite 200, UCRC-II, 11001 Cedar Avenue, in Table 2. All four types of transversions were Cleveland, Ohio 44106, USA recovered, but the A : T?T : A transversion (only Current addresses: 6 Department of Pathology, Ross 632; Johns mutant #1) is underrepresented (P=0.024, binomial Hopkins Medical Institutions, 720 Rutland Avenue, Baltimore, distribution). The three other types of transversions are 7 Maryland 21205; Department of Pathology, University of about equally represented, although the C : G?G:C Kentucky Medical Center, Lexington, Kentucky, 40536, USA Received 18 June 1997; revised 7 October 1997; accepted 7 October transversion occurs most frequently (40% of all 1997 transversions). A colon cancer with a novel transversion mutator JR Eshleman et al 1126 Inspection of the sequences surrounding the mutated an A:T base pair (e.g. mutant #1), and is adjacent to the base suggests that runs of adenine and thymidine mutated base in ten of the 14 mutants involving a G : C nucleotides (Table 2, characters in italics) are found base pair (e.g. mutant #8). To test for signi®cance, all adjacent to most transversions. When de®ned as three transversion sites previously reported as selectable in or more consecutive A or T bases, this motif is present 6TG (Cariello, 1994) were analysed for this A/T motif. in 15 out of 20 transversions. The A/T run includes the This analysis demonstrates that 50% (92 out of 184) of mutated base in ®ve of the six transversions involving the phenotypically selectable transversion sites are ¯anked by three or more consecutive A or T bases. In contrast, 75% (15 out of 20) of the Vaco411 Table 1 Overall distribution of Vaco411 mutations transversions displayed this motif. The dierence Number of Percent of total between sites in the database at which transversions Mutation type mutants mutants (%) have been selected and those found in Vaco411 is Total base substitutions 28 70 statistically signi®cant (P=0.021, binomial distribu- Transversions 20 50 (71)a tion). A search for other motifs surrounding transver- Transitions 8 20 (29)a Small deletions (1 ± 4 bp) 2 5 sion sites in Vaco411 using the MacVector DNA Complex small mutants 2 5 software program (Oxford molecular group, version Large deletions 8 20 5.0) was uninformative. The largest numbers of a Values in parentheses are the percent of transversions or transitions transversions were found at position 403 where two out of all base substitutions C:G?G : C transversions and one C : G?A : T trans- Table 2 Distribution of transversions in the hprt gene of Vaco411 Location of mutation Mutant # Mutation Sequence contexta Siteb Eect 1 A:T?T:A CGTTATGGCGA 2 Met?Lys 2 A:T?C:G TCCTCATGGAC 116 His?Pro 3 A:T?C:G TTGATTGTGGA 396 Ile?Met 4 A:T?C:G GTGAAAAGGAC 498 Lys?Asn 5 A:T?C:G CAAAATACAAA 646 Tyr?Asp 6 A:T?C:G E7-GACT|GTAAGTG-I7 (39,864) D Exon 7 7 C:G?G:C AACCAGGTTAT 46 Gly?Arg 8 C:G?G:C CATACCTAATC 74 Pro?Arg 9 C:G?G:C TCATGGACTAA 119 Gly?Ala 10 C:G?G:C TGCTCGAGATG 152 Arg?Pro 11 C:G?G:C I5-GAAAG|GATATAATTG-E6 403 Asp?His 12 C:G?G:C I5-GAAAG|GATATAATTG-E6 403 Asp?His 13 C:G?G:C GAAAAGGACCC 500 Arg?Thr 14 C:G?G:C GAAAAGCAAAA 640 Ala?Pro 15 C:G?A:T CTGTAGATTTTATC 292 Asp?Tyr 16 C:G?A:T GTCTTGATTGT 393 Leu?Phe 17 C:G?A:T I5-GAAAG|GATATAATTG-E6 403 Asp?Tyr 18 C:G?A:T TCAGGGATTTG 601 Asp?Tyr 19 C:G?A:T I2-CTGTAG|GACTG-E3 (16,602) D E3, D E2-3 20 C:G?A:T E7-T|GTAAGTGAAT-I7 (39,867) D Exon 7 a Sequence context is shown on the sense strand only. The mutated base is depicted in bold and is underlined. Ten surrounding bases are generally depicted unless the adjacent sequence involves an A/T run (italic characters). Introns (I), exons (E), and junctions (|) are designated where necessary. b Site of mutation is generally identi®ed as the cDNA base position. Splice site mutations occurring in introns are designated as genomic DNA base positions depicted in parentheses. Mutants 11, 12, and 17 occurring at position 403 are presented in bold Table 3 Transitions, 1 ± 4 bp deletions and complex mutations in Vaco411 Mutant Location of mutation type Mutant # Mutation Sequence context Site Eect Transitions 21 A:T?G:C E2-TATGGACAG|GT-I2 131 Asp?Gly 22 A:T?G:C TGACCTGCTGG 233 Leu?Pro 23 A:T?G:C E7-GCCAGACT|GTA-I7 530 Asp?Gly 24 C:G?T:A E2-GGACAG|GTAAG-I2 134 Arg?Lys 25 C:G?T:A TTGGTGGAGAT 355 Gly?Arg 26 C:G?T:A GTATAATCCAAAG 463 Pro?Ser 27 C:G?T:A ATAAGCCAGAC 526 Pro?Ser 28 C:G?T:A I8-TATAG|CATGTT-E9 610 His?Tyr 1±4 bp 29 D1 bp ATATGCCCTTGACc 575-577 Frameshift Deletions 30 D3bp I3-TAACTAG|AATGACC-E4c (27890-93) DExon 4 Complex 31 D1 and D4bp CCGCAGCCCTGGCGTCGT 14, 18-21 Frameshift 32 T?A, G?A TTCCTTGGTCAGG 446, 448 Leu?Stop See legend to Table 2. c Denotes the DNA sequence where the small deletions reside in mutants #29 and #30. For these two mutants, an exact identi®cation of the deleted base(s), within the designated area, cannot be made A colon cancer with a novel transversion mutator JR Eshleman et al 1127 version were recovered.